Cloning,Prokaryotic Expression And Enzymatic Characteristics Of The Glutathione-S-Transferase GmolGST5 And GmolGST6 In Grapholita Molesta Busck

Glutathione-S-transferases?GSTs,E.C.2.5.1.18?,a kind of conservatived and multifunctional superfamily enzyme systems,have been found widely exsisting in animals,plants and other aerobic organisms.The main function of GSTs is to catalyze endogenous and exogenous toxic substances to form less-toxic and easier soluble compounds for reducig or avoiding damage to organism.Grapholita molesta Busck is one of main fruit tree pests,which mainly affects rosaceae plants through fruit or branch by larvae.G.molesta has a habit of transfer host damage.This habit has become a significant factor restricting the development of apple,pear,peach and other fruit trees.In our study,we cloned,prokaryotic expressed and preliminarily analyzed the function of glutathione-S-transferase in G.molesta.It is helpful to comprehend the communication mechanism of G.molesta antennae and lay the theoretical basis so as to further study for the new control technologies.The key results of our study are as below.Based on the transcriptome data of female antennae from G.molesta,we selected two glutathione-S-transferase genes,GmolGST5 and GmolGST6.GmolGST5 included 615 bp ORF,which encodes a protein of 204 amino acids.Predicted molecular weight of GmolGST5is 23.29 kD and the theoretical point is 5.78.GmolGST6 included 645 bp ORF,which encodes a protein of 214 amino acids.Predicted molecular weight of GmolGST5 is 24.21 kD and the theoretical point is 5.10.Both of them have no transmembrane regions,and belong to the cytoplasmic GSTs.Sequence alignment and evolutionary tree analysis indicate that GmolGST5,GmolGST6 belong to Sigma,Delta class,respectively.The expression patterns in different tissues of GmolGST5 and GmolGST6 were measured by qPCR.The results showed that GmolGST5 and GmolGST6 expressed in all tissues of both sexes.GmolGST5 expressed the highest in male antennae,followed by the wing.The expression profiles of female in wings is significantly higher than that in male.GmolGST6expressed the highest in male antennae,and is significantly higher compared with other tissues.The expression level of abdomen and wing in female is significantly higher than male.There is no significantly difference in head and thorax of both sexes.Use of prokaryotic expression and protein purification methods,we obtained the soluble protein of about 27 kD.The protein concentration of GmolGST5 and GmolGST6 were 1.16mg/mL and 0.28 mg/mL,respectively.SDS-PAGE and Western-blot indicated that purified protein showed a single band.CDNB is a class substrate for examing the detoxification activity of GSTs.Both of them showed catalyze to CDNB.And the Michaelis constant?K_m?of the recombinant GmolGST5 is 0.21±0.06 mmol/L and the maximum reaction rate(V_max)GmolGST5 is 11.69±0.72?mol/min/mg.The K_m and V_maxax of GmolGST6 is 0.21±0.06mmol/L and 11.69±0.72?mol/min/mg,respectively.Different pH and temperatures have similar effects on the recombinant proteins activity of GmolGST5 and GmolGST6,and both of them first increase and then decrease with increasing pH and temperatures.The optimal reaction pH of both are around pH 7.5.The recombinant protein of GmolGST5 was highly active when it was around 50?,while GmolGST6 was 40?.